The proposed research is designed to elucidate the enzymatic mechanism of biosynthesis of the lipid A region of the lipopolysaccharide of gram-negative bacteria and the mechanism of assembly of the outer membrane of which the lipopolysaccharide is a major constituent. Studies on the biosynthesis of lipid A will emphasize isolation and characterization of mutants conditionally defective in early steps of the pathway, and will focus on biochemical and genetic identification of enzymatic reactions involved in formation of the disaccharaide backbone and its N- and O-acylation by beta-hydroxymyristate, laurate and myristate. Investigations on outer membrane assembly will focus on mechanism of translocation and integration of lipid and protein constituents into the membrane, and the role of lipopolysaccharide and proteins in assembly. Major approaches will include genetic manipulation of outer membrane lipopolysaccharide and protein composition, incorporation of exogenous phospholipid lipopolysaccharide and protein composition, incorporation of exogenous phospholipid lipopolysaccharide and proteins into the outer membrane by a newly developed technique for fusion of intact cells with bilayer phospholipid vesicles, and isolation of lipopolysaccharide binding proteins.